Quantifying three‐dimensional morphology and RNA from individual embryos

Quantitative analysis of morphogenesis aids our understanding of developmental processes by providing a method to link changes in shape with cellular and molecular processes. Over the last decade, many methods have been developed for 3D imaging of embryos using microCT scanning to quantify the shape of embryos during development. These methods generally involve a powerful, cross‐linking fixative such as paraformaldehyde to limit shrinkage during the CT scan. However, the extended time frames that these embryos are incubated in such fixatives prevent use of the tissues for molecular analysis after microCT scanning. This is a significant problem because it limits the ability to correlate variation in molecular data with morphology at the level of individual embryos. Here we outline a novel method that allows RNA, DNA, or protein isolation following CT scan while also allowing imaging of different tissue layers within the developing embryo. We show shape differences early in craniofacial development (E11.5) between common mouse genetic backgrounds, and demonstrate that we are able to generate RNA from these embryos after CT scanning that is suitable for downstream real time PCR (RT‐PCR) and RNAseq analyses. Developmental Dynamics 246:431–436, 2017. © 2017 Wiley Periodicals, Inc. Key Findings This paper presents the first method, to our knowledge, for generating both 3D morphology and RNA. RNA from this method is suitable for downstream analysis such as RNAseq. The combination of morphology and RNA analysis in parallel will allow analysis of a 1:1 correspondence between shape and gene expression.